Spinach plants that are resistant to downy mildew

ABSTRACT

The present invention relates to the field of spinach breeding, in particular to a new dominant resistance gene, designated RPF12, which confers resistance against all races of  Peronospora farinosa  and to spinach plants comprising said gene.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.14/759,957, filed on Jul. 9, 2015, which is the U.S. national phaseentry of PCT application PCT/EP2014/069367, filed Sep. 11, 2014, whichclaims priority to European Patent Application No. 13184393.0, filedSep. 13, 2013, the contents of each of which are hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to a new dominant resistance gene, designatedRPF12, for use in breeding downy mildew resistant spinach plants. Thepresent invention further relates to cultivated spinach (Spinaciaoleracea) seeds, plants and plant parts (e.g. leaves) grown from theseeds, that are resistant to Peronospora farinosa fsp. spinaciae(abbreviated herein as Pfs) due to the presence of RPF12 in theirgenome, as well as to progenies of the plants and propagation materialfor producing the plants.

BACKGROUND OF THE INVENTION

Spinach (Spinacia oleracea) has become an important vegetable crop inmany parts of the world, with the top spinach producing county beingChina (>80% of global production), followed by USA, Japan and variousEuropean countries. Globally about 1 million ha of spinach are grown inAsia and about 35,000 ha in each of the EU, USA and Japan (see Correllet al. (2011, Eur J Plant Pathol 129: 193-205). Part of the increase inspinach demand is likely due to an increased health-consciousness ofconsumers and the beneficial properties of spinach. Spinach leaves arerich in beta-carotene, lutein, folic acid, vitamin C, calcium, iron andantioxidants. Especially the demand for fresh spinach has significantlyincreased over the last years.

Due to this increase in production over the last 10-15 years, incidenceand severity of one of the most damaging pathogens of spinach, downymildew of spinach, caused by races of the oomycete Peronospora farinosafsp. spinaciae (Pfs; synonym P. effusa) has increased concomitantly.Before 1990 only three races of Pfs were known, however between 1990 and2010 ten new races were identified. The emergence of new races of Pfsmakes this pathogen a major threat for spinach production globally andidentifying new sources of resistance is therefore necessary.

Historically, Pfs race 1 (Pfs: 01 or Pfs1) was first reported in 1824and resistance to race 1 was identified later in two Iranian accessions(PI140467 and PI140464) and incorporated into commercial hybridvarieties, such as Califlay (Smith and Zahara, California Agriculture,July 1956). In 1958, race 2 appeared and a few years later a singledominant gene imparting resistance against both race 1 and race 2 wasidentified (Smith et al. 1961 and 1962). In 1976, race 3 appeared andagain several years later resistance against race 3 was found. Race 4was only identified in 1990, and Brandenberger et al. (1992) identifiedaccessions CGN09546, of which 60% of individual plants were resistant,and SP1 82/87, of which 80% of individual plants were resistant. Therapid emergence of new races hereafter, lead to the identification ofnew resistance genes and their incorporation into commercial varieties,as indicated in Table 1 below (− means resistant reaction; + meanssusceptible reaction; (−) means reduced level of infection oftenreferred to as field resistance, i.e. sparse sporulation on the tips ofthe cotyledons; +/− means undecided). These varieties are also used ashost differentials for determining the race of isolates of Pfs (seedocument of the International Seed Federation, August 2013; world wideweb at worldseed.org/isf/differential_hosts.html).

TABLE 1 Variety Pfs1 Pfs2 Pfs3 Pfs4 Pfs5 Pfs6 Pfs7 Pfs8 Pfs9 Pfs10 Pfs11Pfs12 Pfs13 Pfs14 Viroflay + + + + + + + + + + + + + + Resistoflay −− + + + + + + + + + + + + Califlay − + − + − + + − − + − − + − Clermont− − − − + + + + + + + + + + Campania − − − − − + − + + + − + +/− +Boeing − − − − − − − + − + − + − + (=Avenger in USA) Lion − − − − − − −− − + − − − − Lazio − − − − − − − − − − + + + + Whale − − − (−) − (−)(−) − − + − − + (−)

Commercial spinach varieties are mostly hybrids, produced by crossing amale and a female inbred line, although also some open pollinatedvarieties exist. The male and female parent line generally each carry adifferent resistance gene. For example, the hybrid variety Andromeda(Nunhems; see patent application US2012/0222147) is resistant againstPfs 1-12 and Pfs14. Resistance against Pfs1, 3, 5, 8, 9, 11, 12 and 14is conferred by a resistance gene from one parent, while resistanceagainst Pfs 1-10 is conferred by a resistance gene from the otherparent.

WO2013/064436 describes a dominant resistance gene, called R6, whichconfers resistance against Pfs1-6, 9, 11-14 (see Table 1 on page 19 ofWO2013/064436; in 2011 the type strain UA4410 has been designated Pfs14by the International Working Group on Peronospora farinosa, IWGP).

To achieve resistance against all known Pfs races (Pfs1-14) using theknown resistance genes, one would for example need to combine theresistance genes of Lazio and Lion. To date no single resistance gene isknown, which confers resistance against all known Pfs races, orespecially against Pfs races 7-14.

SUMMARY OF VARIOUS EMBODIMENTS OF THE INVENTION

It is, therefore, an object of the invention to provide a singledominant resistance gene, which confers resistance against at least Pfsraces 7-14. In addition, the gene optionally also confers resistanceagainst one or more or all of Pfs races 1-6. Optionally the gene alsoconfers resistance against isolate UA4712. Thus, in one aspect, theresistance gene, designated RPF12, confers resistance against at leastPfs races 7-14, and optionally also against isolate UA4712 (Pfs race 15)and/or optionally against one or more or all of Pfs 1-6 (i.e. againstPfs 1, 2, 3, 4, 5 and 6). In a further aspect, seeds, plants and plantparts or cultivated spinach comprising the new major resistance gene areprovided.

In one aspect, the RPF12 gene is located in between DNA markers SNP_01and SNP_02. Thus, in one aspect, the seed, plant or plant part comprisesin its genome a recombinant chromosome comprising an introgressionfragment from a donor plant in its genome and wherein the introgressionfragment carries the dominant RPF12 gene which is located between SNP_01and SNP_02.

Also methods for identifying and/or selecting spinach plants or plantparts comprising the resistance gene are provided, as are methods fortransferring the resistance gene from seeds deposited under accessionnumber NCIMB 42159 into different spinach plant lines or varieties.

It is a further object to provide one or more markers, especially SNP_01and SNP_02 and markers located in between SNP_01 and SNP_02, that can beused in the selection of plants or plant parts comprising the resistancegene, designated RPF12, which confers resistance against at least Pfsraces 7-14, and optionally also against isolate UA4712 (Pfs race 15)and/or optionally against one or more of Pfs 1-6. Also, methods foreither generating or for identifying plants or plant parts comprisingsaid resistance are provided. In some aspects, methods for selecting,identifying, and/or detecting the resistance gene, designated RPF12,comprise hybridizing one or more nucleic acid probes to a nucleic acidof a plant suspected of comprising RPF12, or amplifying a nucleic acidof a plant suspected of comprising RPF12 using one or more nucleic acidprimers, are provided.

RPF12 is introgressed from wild spinach or a wild relative of spinach(the donor or resistance gene donor) into cultivated spinach (alsoreferred to as the recurrent parent). In one aspect, a cultivatedspinach plant or plant part is provided comprising an introgressionfragment from a wild spinach or wild relative of spinach, wherein theintrogression fragment comprises the dominant RPF12 gene, and whereinthe RPF12 gene is located in between DNA markers SNP_01 and SNP_02.

Also provided is the use of the gene and molecular markers (especiallySingle Nucleotide Polymorphisms or SNPs, especially SNP_01 and/orSNP_02) flanking the gene for the identification of plants or plantparts comprising RPF12, and methods of using such markers in identifyingor generating plants or plant parts comprising RPF12.

In one aspect, the cultivated spinach comprises a recombinantchromosome, said chromosome comprises the introgression fragment whichcomprises RPF12, wherein the recombinant chromosome is the chromosome onwhich SNP_01 and SNP_02 are located. In a further aspect, the remainingchromosomes of the genome of the plant are cultivated spinachchromosomes. In one embodiment, the introgression fragment comprisingthe RPF12 gene is on Linkage Group 1. Without limiting the invention, itis believed that Linkage Group 1 corresponds to Linkage Group P03 ofChan-Navarrete et al. (Euphytica 2016, 208:621-636).

In one aspect, the RPF12 gene is the gene present in seeds depositedunder accession number NCIMB 42159 or in progeny thereof which retainthe RPF12 gene in their genome, such as progeny which retain the RPF12gene located in between SNP_01 and SNP_02. In one aspect, the progenyretain the SNP_01 and/or SNP_02 genotype of the donor, although theskilled person can also select plants which retain the RPF12 gene butlack SNP_01 and/or SNP_02 of the donor and thus comprise a shorterintrogression fragment. Thus, in one aspect, the SNP genotype of SNP_01and/or SNP_02 may also be the genotype of the recurrent parent, whilethe RPF12 gene is still present. A shorter introgression fragment can beidentified by markers in between SNP_01 and SNP_02 which have a donorgenotype and are linked to the RPF12 gene. The distance between SNP_01and SNP_02 is 45.949 bases (nucleotides), i.e. 45.949 nucleotides liebetween SNP_01 and SNP_02. Thus RPF12 lies in the region starting atSNP_01 and ending at SNP_02 and sub-regions comprising the RPF12 genecan be identified e.g. by sequencing the region or by fine-mapping.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION Definitions

The indefinite article “a” or “an” does not exclude the possibility thatmore than one of the element is present, unless the context clearlyrequires that there be one and only one of the elements. The indefinitearticle “a” or “an” thus usually means “at least one”.

“Plant variety” is a group of plants within the same botanical taxon ofthe lowest grade known, which (irrespective of whether the conditionsfor the recognition of plant breeder's rights are fulfilled or not) canbe defined on the basis of the expression of characteristics that resultfrom a certain genotype or a combination of genotypes, can bedistinguished from any other group of plants by the expression of atleast one of those characteristics, and can be regarded as an entity,because it can be multiplied without any change. Therefore, the term“plant variety” cannot be used to denote a group of plants, even if theyare of the same kind, if they are all characterized by the presence ofone or two loci or genes (or phenotypic characteristics due to thesespecific loci or genes), but which can otherwise differ from one anotherenormously as regards the other loci or genes.

“Spinach” or “cultivated spinach” or “cultivated Spinacia oleracea”refers herein to plants of the species Spinacia oleracea (or seeds fromwhich the plants can be grown), and parts of such plants, bred by humansfor food and having good agronomic characteristics. This includes anycultivated spinach, such as breeding lines (e.g. backcross lines, inbredlines), cultivars and varieties (open pollinated or hybrids). Thisincludes any type of spinach, such as savoy, flat- or smooth-leafspinach or semi-savoy types. Wild spinach (i.e. not cultivated spinach)or wild relatives of spinach, such as Spinacia tetrandra and Spinaciaturkestanica, are not encompassed by this definition.

“Wild spinach” or “wild relatives of spinach” refer herein to thespecies Spinacia tetrandra and/or Spinacia turkestanica. These speciesare also referred to as the donor plants or donor species of the RPF12gene, i.e., the introgression fragment comprising RPF12 is derived fromsuch a wild spinach donor plant. The cultivated spinach into which theRPF12 gene of the donor is introgressed is referred to as the recurrentparent or cultivated parent. In one aspect the RPF12 donor plant is ofthe species Spinacia turkestanica.

As used herein, the term “plant” includes the seed (from which the plantcan be grown), the whole plant or any parts such as plant organs (e.g.,harvested or non-harvested leaves, etc.), plant cells, plantprotoplasts, plant cell- or tissue cultures from which whole plants canbe regenerated, propagating or non-propagating plant cells, plants cellswhich are not in tissue culture (but which are for example in vivo in aplant or plant part), plant callus, plant cell clumps, planttransplants, seedlings, plant cells that are intact in plants, plantclones or micro-propagations, or parts of plants (e.g., harvestedtissues or organs), such as plant cuttings, vegetative propagations,embryos, pollen, ovules, flowers, leaves, heads, seeds (produced on theplant after self-fertilization or cross-fertilization), clonallypropagated plants, roots, stems, stalks, root tips, grafts, parts of anyof these and the like, or derivatives thereof, preferably having thesame genetic make-up (or very similar genetic make-up) as the plant fromwhich it is obtained. Also any developmental stage is included, such asseedlings, cuttings prior or after rooting, mature and/or immatureplants or mature and/or immature leaves. When “seeds of a plant” arereferred to, these either refer to seeds from which the plant can begrown or to seeds produced on the plant, after self-fertilization orcross-fertilization.

“Somatic cells” and “reproductive cells” can be distinguished, wherebysomatic cells are cells other than gametes (e.g. ovules and pollen),germ cells and gametocytes. Gametes, germ cells and gametocytes are“reproductive cells”.

“Tissue Culture” or “cell culture” refers to an in vitro compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into plant tissue. Tissue culturesand cell cultures of spinach, and regeneration of spinach plantstherefrom, is well known and widely published (see, e.g. Nguyen et al.,2013, Plant Biotechnology Reports, Vol. 7 Issue 1, p 99).

“Harvested plant material” refers herein to plant parts (e.g., leavesdetached from the whole plant) which have been collected for furtherstorage and/or further use.

“Harvested seeds” refers to seeds harvested from a line or variety,e.g., produced after self-fertilization or cross-fertilization andcollected.

“Harvested leaves” as used herein refers to spinach leaves, i.e., theplant without the root system, for example substantially all (harvested)leaves.

“Progeny” or “progenies” or “descendants” as used herein refers tooffspring, or the first and all further descendants derived from(obtainable from) (derivable from or obtained from) a plant of theinvention that comprises (retains) the RPF12 resistance gene inhomozygous or heterozygous form and/or the RPF12 resistance phenotypedescribed herein. Progeny may be derived by regeneration of cell cultureor tissue culture, or parts of a plant, or selfing of a plant, or byproducing seeds of a plant. In further embodiments, progeny may alsoencompass spinach plants derived from crossing of at least one spinachplant with another spinach plant of the same or another variety or(breeding) line, and/or backcrossing, and/or inserting of a locus into aplant and/or mutation. A progeny is, e.g., a first generation progeny,i.e. the progeny is directly derived from, obtained from, obtainablefrom or derivable from the parent plant by, e.g., traditional breedingmethods (selfing and/or crossing) or regeneration. However, the term“progeny” generally encompasses further generations such as second,third, fourth, fifth, sixth, seventh or more generations, i.e.,generations of plants which are derived from, obtained from, obtainablefrom or derivable from the former generation by, e.g., traditionalbreeding methods, regeneration or genetic transformation techniques. Forexample, a second generation progeny can be produced from a firstgeneration progeny by any of the methods mentioned above. Also doublehaploid plants are progeny.

“Plant line” is for example a breeding line which can be used to developone or more varieties. “Inbred line” or “inbred parent” is a line whichhas been developed by selfing for several generations and which can beused as a parent to produce an F1 hybrid variety.

“F1, F2, F3, etc.” refers to the consecutive related generationsfollowing a cross between two parent plants or parent lines. The plantsgrown from the seeds produced by crossing two plants or lines is calledthe F1 generation. Selfing the F1 plants results in the F2 generation,etc.

“Hybrid” refers to the seeds harvested from crossing one plant line orvariety with another plant line or variety, and the plants or plantparts grown from said seeds.

“F1 hybrid” plant (or F1 hybrid seed) is the generation obtained fromcrossing two non-isogenic inbred parent lines. Thus, F1 hybrid seeds areseeds from which F1 hybrid plants grow.

An “interspecific hybrid” refers to a hybrid produced from crossing aplant of one species, e.g. S. oleracia, with a plant of another species,e.g. S. tetrandra or S. turkestanica.

“Crossing” refers to the mating of two parent plants. Equally“Cross-pollination” refers to fertilization by the union of two gametesfrom different plants.

“Selfing” refers to the self-pollination of a plant, i.e. to the unionof gametes from the same plant.

“Backcrossing” refers to a breeding method by which a (single) trait,such as Pfs resistance conferred by the RPF12 resistance gene, can betransferred from one genetic background (also referred to as “donor”;generally but not necessarily this is an inferior genetic background)into another genetic background (also referred to as “recurrent parent”;generally but not necessarily this is a superior genetic background). Anoffspring of a cross (e.g. an F1 plant obtained by crossing a wildspinach or wild relative of spinach with a cultivated spinach; or an F2plant or F3 plant, etc., obtained from selfing the F1) is “backcrossed”to the parent with the superior genetic background, e.g. to thecultivated parent. After repeated backcrossing, the trait of the donorgenetic background, e.g. the RPF12 gene, will have been incorporatedinto the recurrent genetic background. The terms “gene converted” or“conversion plant” or “single locus conversion” in this context refer toplants which are developed by backcrossing wherein essentially all ofthe desired morphological and/or physiological characteristics of therecurrent parent are recovered in addition to the one or more genes(e.g. the RPF12 resistance gene) transferred from the donor parent.

The term “traditional breeding techniques” encompasses herein crossing,backcrossing, selfing, selection, chromosome doubling, double haploidproduction, embryo rescue, the use of bridge species, protoplast fusion,marker assisted selection, mutation breeding etc. as known to thebreeder (i.e. methods other than geneticmodification/transformation/transgenic methods), by which, for example,the RPF12-resistance gene can be obtained, identified, selected, and/ortransferred.

“Regeneration” refers to the development of a plant from in vitro cellculture or tissue culture or vegetative propagation.

“Vegetative propagation”, “vegetative reproduction” or “clonalpropagation” are used interchangeably herein and mean the method oftaking part of a plant and allowing that plant part to form at leastroots where plant part is, e.g., defined as or derived from (e.g. bycutting off) leaf, pollen, embryo, cotyledon, hypocotyl, cells,protoplasts, meristematic cell, root, root tip, pistil, anther, flower,shoot tip, shoot, stem, fruit, and petiole. When a whole plant isregenerated by vegetative propagation, it is also referred to as a“vegetative propagation” or a “vegetatively propagated plant”.

“Single locus converted (conversion) plant” refers to plants which aredeveloped by plant breeding techniques comprising or consisting ofbackcrossing, wherein essentially all of the desired morphologicaland/or physiological characteristics of a spinach plant are recovered inaddition to the characteristics of the single locus (e.g. the locuscomprising the RPF12 gene) having been transferred into the plant viathe backcrossing technique and/or by genetic transformation.

“Transgene” or “chimeric gene” refers to a genetic locus comprising aDNA sequence which has been introduced into the genome of a spinachplant by transformation. A plant comprising a transgene stablyintegrated into its genome is referred to as “transgenic plant”.

“Pfs” or “Peronospora farinosa” or “downy mildew” refers to races of theoomycete Peronospora farinosa fsp. spinaciae. Pfs1-Pfs14 refer to theofficially recognized races, which can be differentiated on thedifferential hosts of spinach and which can be obtained from theNaktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands, orvia references provided by the ISF (International Seed Federation).

“Differential hosts” or “differentials” refers to the differential hostsof spinach for distinguishing Pfs races 1-14, which can be obtained fromthe Naktuinbouw, P.O. Box 40, 2370 AA Roelofarendsveen, The Netherlands,or via references provided by the ISF (International Seed Federation).

“UA4712” refers to a pathogenic isolate of Pfs which has by now beenofficially recognized as a new Pfs race, Pfs15. It was originallydescribed by Correl and Koike, 2013, Race diversity and the biology ofthe spinach downy mildew pathogen, CLGRB Annual Report, Apr. 1, 2012 toMar. 31, 2013. The UA4712 isolate was first identified in November 2012in Imperial Valley, Calif., U.S. The isolate was evaluated for diseasedevelopment in a test against a standard set of differential varieties,and as of September 2014 the International Working Group on Peronospora(IWGP) determined that the isolate was a new race, and named it Pfs 15once it became clear that isolates with the same reaction patternoccurred in many locations (Plantum press release 2 Sep. 2014“Denomination of Pfs: 15, a new race of downy mildew in spinach”). Inthis application, UA4712, Pfs 15, Pfs race 15 and Pfs: 15 will be usedinterchangeably.

A “Pfs resistant plant” or “downy mildew resistant plant” or a planthaving “Pfs resistance” or a “Pfs resistant phenotype” refers to aspinach plant which is resistant against one or more pathogenic races(and pathogenic isolates) of Pfs, as determined in a qualitativeresistance assay under controlled environmental conditions. In such aresistance assay a plurality of plants (e.g. at least 2 replicates of atleast 10 plants) of a genotype, are inoculated with a sporangialsuspension of the race or isolate and incubated under suitableconditions. After a suitable incubation period (e.g. 7, 8, 9, 10, 11 ormore days after inoculation) the plants are evaluated for symptoms.Susceptible controls should show sporulation at the time of symptomevaluation. Any plant showing sporulation on the cotyledons (and/or onthe true leaf/leaves) is considered “susceptible”, while any plant notshowing any sporulation on the cotyledons (and/or on the trueleaf/leaves) is considered “resistant”. A plant genotype with more than85% of the inoculated plants (preferably more than 95%) being classifiedas “resistant” plant is considered to a resistant against the race orisolate. In the test >85% of inoculated plants (preferably more than 95%of plants) of the susceptible control plant, such as cultivar Viroflay,should show sporulation. Suitable tests are described herein in theExamples, or in Irish et al. 2007 (Plant Disease Vol 91 No. 11, inMaterials and Methods on page 1392-1394), or in Correll et al. 2010,“Guidelines for Spinach Downy Mildew: Peronspora ferinosa fsp. spinaciae(Pfs)” found on the website of the ISF (International Seed Federation).

“RPF12” refers herein to a single, dominant Pfs resistance gene, whichconfers Pfs resistance (as defined above) against at least races 7-14,and optionally against UA4712 (and optionally against new pathogenicisolates). In one embodiment RPF12 refers to a resistance gene whichconfers resistance against at least races 7-14 and optionally furtheragainst one or more or all of races 1-6, and/or optionally againstUA4712 (and optionally against new pathogenic isolates). The resistanceagainst races 1 to 6 and 9 to 15 is conferred when the gene is inhomozygous or heterozygous form and the resistance against races 7 and 8is conferred when the gene is in homozygous form. The resistancephenotype is also referred to herein as the “Pfs resistance phenotypeconferred by the RPF12 gene”. In one embodiment, the RPF12 gene islocated in between markers SNP_01 and SNP_02. The term “locus” (lociplural) means a specific place or places or a site on a chromosomewhere, for example, a gene (e.g. the RPF12 gene) or genetic marker isfound. In spinach according to the invention, the resistance locuscomprising the RPF12 gene is introgressed from a wild spinach or wildrelative of spinach (i.e. the donor plant) into cultivated spinach (S.oleracea).

The term “locus” (loci plural) means a specific place or places or asite on a chromosome where for example a gene (e.g. the RPF12 gene) orgenetic marker is found. In spinach according to the invention theresistance locus comprising the RPF12 gene is introgressed from a wildspinach or wild relative of spinach (i.e. the donor plant) intocultivated spinach (S. oleracea).

The term “allele(s)” means any of one or more alternative forms of agene at a particular locus, all of which alleles relate to one trait orcharacteristic at a specific locus. In a diploid cell of an organism,alleles of a given gene are located at a specific location, or locus(loci plural) on a chromosome. One allele is present on each chromosomeof the pair of homologous chromosomes. A diploid plant species maycomprise a large number of different alleles at a particular locus.These may be identical alleles of the gene (homozygous) or two differentalleles (heterozygous).

The term “gene” means a (genomic) DNA sequence comprising a region(transcribed region), which is transcribed into a messenger RNA molecule(mRNA) in a cell, and an operably linked regulatory region (e.g. apromoter). Different alleles of a gene are thus different alternativeforms of the gene, which may be in the form of e.g. differences in oneor more nucleotides of the genomic DNA sequence (e.g. in the promotersequence, the exon sequences, intron sequences, etc.), mRNA and/or aminoacid sequence of the encoded protein.

“Allelism test” refers to a genetic test whereby it can be testedwhether a phenotype, such as Pfs resistance, seen in two plants, isdetermined by the same gene or by different genes. For example, theplants to be tested are crossed with each other, the F1 is selfed andthe segregation of the phenotypes amongst the F2 progeny is determined.Other segregating populations can equally be made (e.g. backcrosspopulations). The ratio of segregation of the phenotype indicates if thegenes are allelic (alleles of the same gene) or non-allelic (different,independent genes).

“Introgression fragment” or “introgression segment” or “introgressionregion” refers to a chromosome fragment (or chromosome part or region)which has been introduced into another plant of the same or relatedspecies by crossing or traditional breeding techniques, such asbackcrossing, i.e. the introgressed fragment is the result of breedingmethods referred to by the verb “to introgress” (such as backcrossing).In spinach, wild spinach or wild relatives of spinach are used tointrogress fragments of the wild genome into the genome of cultivatedspinach. Such a spinach plant thus has a “genome of Spinacia oleracea”,but comprises in the genome a fragment of a wild spinach or spinachrelative, i.e. an introgression fragment of a donor plant. It isunderstood that the term “introgression fragment” never includes a wholechromosome, but only a part of a chromosome. The introgression fragmentcan be large, e.g. even half of a chromosome, but is preferably smaller,such as about 15 Mb or less, such as about 10 Mb or less, about 9 Mb orless, about 8 Mb or less, about 7 Mb or less, about 6 Mb or less, about5 Mb or less, about 4 Mb or less, about 3 Mb or less, about 2 Mb orless, about 1.5 Mb or less, about 1 Mb (equals 1,000,000 base pairs) orless, or about 0.5 Mb (equals 500,000 base pairs) or less, such as about200,000 bp (equals 200 kilo base pairs) or less, about 100,000 bp (100kb) or less, about 50,000 bp (50 kb) or less, about 25,000 bp (25 kb) orless.

“RPF12 comprising introgression fragment” or “RPF12 comprisingintrogression segment” or “RPF12 comprising introgression region” refersto the chromosome fragment comprising the RPF12 gene, whereby the RPF12gene is located on the chromosome region in between SNP_01 and SNP_02.SNP_01 refers to nucleotide 101 of SEQ ID NO 1 or of a sequencecomprising at least 90%, 95%, 97%, 98% or 99% sequence identity to SEQID NO: 1. SNP_02 refers to nucleotide 101 of SEQ ID NO 2 of a sequencecomprising at least 90%, 95%, 97%, 98% or 99% sequence identity to SEQID NO: 2.

“SNP genotype” refers to the nucleotide found at the SNP location, i.e.at nucleotide position 101 of SEQ ID NO: 1 for SNP_01 and SEQ ID NO: 2for SNP_02 (or at the corresponding position of a sequence comprising atleast 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 or SEQID NO: 2, respectively). A “resistant SNP genotype” or a “donor SNPgenotype” refers to an Thymine (T) being present at nucleotide 101 ofthe mentioned sequences. A “susceptible SNP genotype” or a “recurrentparent SNP genotype” or “cultivated SNP genotype” means that a Guanine(G), Cytosine (C) or Adenine (A) is present at nucleotide 101 of thementioned sequences.

The same applies for markers located in between SNP_01 and SNP_02, andwhich are linked to the RPF12 gene. Such markers also have a donorgenotype indicative of the introgression fragment or a recurrent parentgenotype indicative of the cultivated spinach.

“Sequence identity” can be determined by alignment of two nucleotidesequences using global or local alignment algorithms. Sequences may thenbe referred to as “substantially identical” or “essentially similar”when they are optimally aligned by for example the programs GAP orBESTFIT or the Emboss program “Needle” (using default parameters, seebelow) share at least a certain minimal percentage of sequence identity(defined further below). These programs use the Needleman and Wunschglobal alignment algorithm to align two sequences over their entirelength, maximizing the number of matches and minimizes the number ofgaps. Generally, the default parameters are used, with a gap creationpenalty=10 and gap extension penalty=0.5 (both for nucleotide andprotein alignments). For nucleotides the default scoring matrix used isDNAFULL. Sequence alignments and scores for percentage sequence identitymay for example be determined using computer programs, such as EMBOSS asavailable on the world wide web underebi.ac.uk/Tools/psa/emboss_needle/). Alternatively sequence similarityor identity may be determined by searching against databases such asFASTA, BLAST, etc., but hits should be retrieved and aligned pairwise tocompare sequence identity. Two nucleic acid sequences have “substantialsequence identity” if the percentage sequence identity is at least 90%,95%, 96%, 97%, 98%, 99% or more (as determined by Emboss “needle” usingdefault parameters, i.e. gap creation penalty=10, gap extensionpenalty=0.5, using scoring matrix DNAFULL for nucleic acids).

“Physical distance” between loci (e.g. between molecular markers and/orbetween phenotypic markers) on the same chromosome is the actualphysical distance expressed in base pairs (bp), kilo base pairs (kb) ormegabase pairs (Mb).

“Genetic distance” between loci (e.g. between molecular markers and/orbetween phenotypic markers) on the same chromosome is measured byfrequency of crossing-over, or recombination frequency (RF) and isindicated in centimorgans (cM). One cM corresponds to a recombinationfrequency of 1%. If no recombinants can be found, the RF is zero and theloci are either extremely close together physically or they areidentical. The further apart two loci are, the higher the RF.

A genetic element, a locus, an introgression fragment or a gene orallele conferring a trait (such as resistance against Pfs) is said to be“obtainable from” or can be “obtained from” or “derivable from” or canbe “derived from” or “as present in” or “as found in” a plant or seed ifit can be transferred from the plant or seed in which it is present intoanother plant or seed in which it is not present (such as a line orvariety) using traditional breeding techniques without resulting in aphenotypic change of the recipient plant apart from the addition of thetrait conferred by the genetic element, locus, introgression fragment,gene or allele. The terms are used interchangeably and the geneticelement, locus, introgression fragment, gene or allele can thus betransferred into any other genetic background lacking the trait. Notonly seeds deposited and comprising the genetic element, locus,introgression fragment, gene or allele can be used, but alsoprogeny/descendants from such seeds which have been selected to retainthe genetic element, locus, introgression fragment, gene or allele, canbe used and are encompassed herein, such as commercial varietiesdeveloped from the deposited seeds or from descendants thereof. Whethera plant comprises the same genetic element, locus, introgressionfragment, gene or allele as obtainable from the deposited seeds can bedetermined by the skilled person using one or more techniques known inthe art, such as phenotypic assays, whole genome sequencing, molecularmarker analysis, trait mapping, chromosome painting, allelism tests andthe like.

A “molecular marker” is a piece of DNA associated with a certain genomicor chromosomal location or single nucleotide polymorphism (SNP), whichis found on the chromosome close to the gene of interest, preferablyclose to RPF12. Molecular markers can be used to identify a particularsequence of DNA, or a certain location in a genome or on a chromosome,or to identify an introgression fragment. When reference is made hereinto one or more molecular markers being “detectable” by a molecularmarker assay, this means of course that the plant or plant partcomprises the one or more markers in its genome, as the marker wouldotherwise not be detectable. In one aspect, the marker is a SingleNucleotide Polymorphism (SNP), but other molecular markers such as RFLP,AFLP, RAPD, INDEL, DNA sequencing, etc. may equally be used. In oneaspect, the marker SNP_01 at position 101 of SEQ ID NO: 1 (or of asequence comprising substantial sequence identity to SEQ ID NO: 1)and/or SNP_02 at position 101 of SEQ ID NO 2 (or of a sequencecomprising substantial sequence identity to SEQ ID NO: 2), or anymolecular marker located in between SNP_01 and SNP_02 and being linkedto the RPF12 gene can be used to select plants, plant tissues or plantparts comprising RPF12, and thus to select and/or generate Pfsresistance (as defined above) against at least races 7-14, andoptionally against UA4712 (AKA Pfs race 15) (and optionally against newpathogenic isolates) or optionally further against one or more of races1-6 (e.g. against at least races 1, 2, 3, 4, 5 and 6).

“Flanking markers” are molecular markers located on the chromosome oneither side of the RPF12 gene. SNP_01 and SNP_02 are flanking markers,i.e. the RPF12 gene is located in between SNP_01 and SNP_02.

Alternatively, other molecular markers can be developed which are linkedto RPF12 and which lie in between SNP_01 and SNP_02 and these can thenbe used for identification or selection of the RPF12 gene, conferringPfs resistance (as defined above) against at least races 7-14, andoptionally against UA4712 (AKA Pfs race 15) (and optionally against newpathogenic isolates) or optionally further against one or more or all ofraces 1-6 (e.g. against races 1, 2, 3, 4, 5 and 6). For example,fine-mapping can be carried out to find markers in the region betweenSNP_01 and SNP_02 which are linked even more closely to the RPF12 gene.Fine mapping involves making recombinant plants, which compriserecombinations in the region between SNP_01 and SNP_02 and analyzingwhether these recombinant plants retain or lose the RPF12 gene. Thereby,the location of the RPF12 gene can be defined more precisely and markersin between SNP_01 and SNP_02 which are linked more closely to the genecan be identified. Alternatively, sequencing can be carried out toidentify markers in-between SNP_01 and SNP_02 closely linked to theRPF12 gene or even within the gene.

The term “marker assay” or “genotyping assay” refers to an assay whichcan be used to determine the marker genotype, e.g. the SNP genotype. Forexample SNP markers can be detected using a KASP-assay (see world wideweb at kpbioscience.co.uk) or other assays known to the skilled person.

“Marker assisted selection” or “MAS” is a process of using the presenceof molecular markers, which are genetically and physically linked to aparticular locus or to a particular chromosome region (e.g.introgression fragment), to select plants (e.g. progeny) for thepresence of the specific locus or region (e.g. introgression fragment).For example SNP_01 and/or SNP_02, or any marker in between SNP_01 andSNP_02, may be used in MAS to select spinach plants or plant partscomprising the RPF12 gene.

When reference is made to a nucleic acid sequence (e.g. DNA or genomicDNA) having “substantial sequence identity to” a reference sequence orhaving a sequence identity of at least 80%, e.g. at least 85%, 90%, 92%,95%, 98%, 99%, 99.2%, 99.5%, 99.9% nucleic acid sequence identity to areference sequence, in one embodiment said nucleotide sequence isconsidered substantially identical to the given nucleotide sequence andcan be identified using stringent hybridisation conditions. In anotherembodiment, the nucleic acid sequence comprises one or more mutationscompared to the given nucleotide sequence but still can be identifiedusing stringent hybridisation conditions.

“Stringent hybridisation conditions” can be used to identify nucleotidesequences, which are substantially identical to a given nucleotidesequence. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequences at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridises to a perfectly matched probe. Typicallystringent conditions will be chosen in which the salt concentration isabout 0.02 molar at pH 7 and the temperature is at least 60° C. Loweringthe salt concentration and/or increasing the temperature increasesstringency. Stringent conditions for RNA-DNA hybridisations (Northernblots using a probe of e.g. 100 nt) are for example those which includeat least one wash in 0.2×SSC at 63° C. for 20 min, or equivalentconditions. Stringent conditions for DNA-DNA hybridisation (Southernblots using a probe of e.g. 100 nt) are for example those which includeat least one wash (usually 2) in 0.2×SSC at a temperature of at least50° C., usually about 55° C., for 20 min, or equivalent conditions. Seealso Sambrook et al. (1989) and Sambrook and Russell (2001).

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is the wild donor sequence for SNP_01 comprising an Thymine(T) at nucleotide position 101, whereas the recurrent parent comprises aCytosine (C) at nucleotide 101.

SEQ ID NO: 2 is the wild donor sequence for SNP_02 comprising a Thymine(T) at nucleotide position 101, whereas the recurrent parent comprises aCytosine (C) at nucleotide 101.

Plants and Methods

The invention provides a spinach plant comprising resistance against atleast Peronospora farinosa races 7-14, wherein said resistance isconferred by a single gene. The gene, designated herein RPF12 (forResistance to Peronospora Farinosa 12) further optionally confersresistance against one or more or all of Peronospora farinosa races 1-6.In one aspect the RPF12 gene, therefore, confers resistance against allcurrently known pathogenic Pfs races, races 1-14, when in homozygous orheterozygous form in the plant. It is noted that resistance againstraces Pfs 7 and 8 is only seen when the gene is in homozygous form. Inanother aspect the RPF12 gene confers resistance against pathogenic Pfsraces, races 1 to 15, when in homozygous form and against races 1 to 6and 9 to 14 when in heterozygous form in the plant. Optionally, theresistance gene also confers resistance against one or more pathogenicisolates of Pfs, such as isolate UA4712 (Pfs race 15) when in homozygousor heterozygous form, or other isolates, including future isolates orraces which develop in the field due to large scale uses of Pfsresistant spinach varieties. Thus, in another aspect the RPF12 geneconfers resistance against at least pathogenic Pfs races 7-14 andagainst UA4712 (race 15), when in homozygous form, or against races 9-14and against UA4712 (race 15) when in heterozygous form, in the plant.

The RPF12 gene is a single, dominant resistance gene, i.e. when a plantcomprising RPF12 in homozygous form (such as a plant grown from seeddeposited under accession number NCIMB 42159) is crossed with asusceptible plant, such as variety Viroflay, the F2 progeny willsegregate in a 3 (resistant): 1 (susceptible) ratio, at least regardingthe resistance against Pfs races 1 to 6 and 9 to 14 and 15.

The RPF12 gene was found to be located in between the molecular markersSNP_01 and SNP_02. Therefore, in one aspect a spinach plant of thespecies Spinacia oleracea is provided comprising resistance againstPeronospora farinosa races 7-14, wherein said resistance is conferred bya single dominant gene introgressed from Spinacia tetrandra or Spinaciaturkestanica, which gene is located between SNP_01 at nucleotide 101 ofSEQ ID NO: 1 or of a sequence comprising at least 95% sequence identityto SEQ ID NO: 1 and SNP_02 at nucleotide 101 of SEQ ID NO: 2 or of asequence comprising at least 95% sequence identity to SEQ ID NO: 2.

In the mapping population, the SNP genotype of the donor was Thymine forSNP_01, rather than Cytosine for the recurrent parent. The SNP genotypeof the donor was Thymine for SNP_02, rather than Cytosine for therecurrent parent. Therefore, in one aspect, the nucleotide of SNP_01and/or of SNP_02 is Thymine. A diploid spinach plant homozygous for theintrogression fragment comprising RPF12 therefore has, in one aspect,the SNP_01 genotype ‘TT’ (one Thymine on each chromosome) and/or theSNP_02 genotype of ‘TT’ (one Thymine on each chromosome). A plantheterozygous for the introgression fragment comprises the SNP_01genotype ‘TC’ (or ‘TG’ or ‘TA’, depending on the recurrent parentbackground) and/or the SNP_02 genotype ‘TC’ (or ‘TG’ or ‘TA’ dependingon the recurrent parent background).

In one embodiment, a spinach plant is encompassed of the speciesSpinacia oleracea comprising resistance against Peronospora farinosaraces 7-14, wherein said resistance is conferred by a single dominantgene introgressed from Spinacia tetrandra or Spinacia turkestanica,which gene is located between SEQ ID NO: 1 or of a sequence comprisingat least 95% sequence identity to SEQ ID NO: 1 and SEQ ID NO: 2 or of asequence comprising at least 95% sequence identity to SEQ ID NO: 2.

The RPF12 gene can be identified in different wild spinach accessions,especially in accessions of the species Spinacia tetrandra or Spinaciaturkestanica, and can be introgressed into cultivated spinach. To dothis, the skilled person can screen wild spinach accessions for thepresence of the resistance genotype for SNP_01 and/or SNP_02 and/or anymarker in between SNP_01 and SNP_02. Alternatively, or in addition, theskilled person can test the Pfs resistance and optionally theinheritance (as single dominant gene) to determine if the wild accessioncontains RPF12. Finally, the skilled person can also do allelism testsor sequence the chromosome region between SNP_01 and SNP_02 to identifythe presence of RPF12.

In one aspect, the RPF12 resistance gene is the gene present in plantsgrown from seeds, a representative sample of seeds having been depositedunder accession number NCIMB 42159. However, as mentioned above, theRPF12 gene may also be derived from a different wild spinach or wildrelative of spinach and introgressed into cultivated spinach.

The cultivated spinach plant according to the invention is, in oneembodiment, a hybrid plant, especially an F1 hybrid made by crossing twoinbred parent lines. The hybrid may comprise the introgression fragmentcomprising the RPF12 gene in homozygous or heterozygous form. In anotherembodiment, the cultivated spinach plant according to the invention isan inbred line. Such an inbred line may be a male or female line. It maycomprise the introgression fragment in homozygous form. The cultivatedspinach plant may be selected from the group consisting of: savoy,semi-savoy, flat- or smooth leaved.

In a specific aspect, the resistance against Peronospora farinosa isconferred by an introgression fragment from Spinacia turkestanica. Thecultivated spinach plant therefore comprises the RPF12 gene locatedbetween SNP_01 and SNP_02, wherein the RPF12 gene is derived fromSpinacia turkestanica.

The introgression fragment is, in one aspect, the fragment as found inspinach seeds, a representative sample of seeds having been depositedunder accession number NCIMB 42159, or a sub-fragment thereof. Asub-fragment is a shorter fragment which still retains the RPF12 gene,but where on either side or on both sides of the gene a part of theintrogression region has been removed by recombination. For example, thedonor genotype of SNP_01 and/or SNP_02 may have been removed byrecombination and is replaced by the cultivated spinach region of thechromosome. The skilled person can easily generate and identify plantscomprising such sub-fragments of the introgression fragment of seedsdeposited under NCIMB42159. The skilled person can, for example, cross aplant grown from the deposited seeds with another cultivated spinachplant (e.g. a plant susceptible to one or more of Pfs races 7-15), andthen self the F1 progeny to produce an F2 population and identifyrecombinants (cross-over events) having occurred in the region betweenSNP_01 and SNP_02. Such sub-fragments contain donor markers, i.e.markers which lie in between SNP_01 and SNP_02.

The single, dominant RPF12 resistance gene, which is effective againstall know pathogenic races, is of great advantage in generating resistantspinach varieties. To date, resistance genes with complementaryresistance phenotypes have been stacked to provide resistance againstseveral races. For example, the F1 hybrid variety Andromeda is a stackof two resistance genes, one inherited from the female parent and onefrom the male parent line. Therefore, the products described herein(e.g. plants, plant parts, progeny plants, etc.) provide a significantimprovement over the prior art.

A representative sample of seeds of a spinach line comprising the RPF12gene in homozygous form has been deposited under Accession number NCIMB42159.

In one aspect of the invention a spinach plant comprising the RPF12resistance gene is obtainable by (or obtained by, or derivable from, orderived from) crossing a spinach plant grown from seeds deposited underaccession number NCIMB 42159, with another spinach plant, for examplewith a spinach plant lacking Pfs resistance genes (a susceptible plant)or with a spinach plant comprising one or more different Pfs resistancegenes. Thus, in one aspect, a spinach plant comprising resistanceagainst at least Peronospora far/nose races 7-14 is provided wherein theresistance is conferred by a single dominant gene, called RPF12, whereinthe RPF12 gene is the gene as present in seeds deposited underNCIMB42159 or progeny thereof.

In one embodiment of the invention, the RPF12 gene is, therefore thegene as found in plants (or plant parts) grown from seeds depositedunder accession number NCIMB 42159 or in progeny of such plants, such asplants obtained by selfing NCIMB 42159 or by crossing NCIMB 42159 withanother spinach plant to obtain progeny and by retaining the resistancegene in the progeny (e.g. using phenotypic and/or molecular methods toidentify or select progeny containing the RPF12 gene). Preferably, saidmolecular methods includes the use of molecular markers SNP_01 and/orSNP_02 and/or any molecular marker located in between SNP_01 and SNP_02and linked to the introgressed RPF12 gene.

Therefore in one embodiment of the invention a spinach plant is providedcomprising resistance against Peronospora farinosa races 7-14, whereinsaid resistance is conferred by a single gene, wherein said gene is thegene as found in (or as obtainable from) seeds deposited under accessionnumber NCIMB42159, e.g. by crossing a plant grown from seeds ofaccession number NCIMB42159 with another spinach plant. In one aspectthe (dominant) gene is in homozygous or heterozygous form. In anotheraspect, the single gene is detectable by the donor genotype of SNP_01and/or SNP_02 and/or any molecular marker located in between SNP_01 andSNP_02 and linked to the introgressed RPF12 gene.

Thus, in one aspect a method for screening, and optionally selecting,spinach seeds, plants or plant parts or DNA from such seeds, plants orplant parts for the presence of one or more markers linked to the RPF12gene is provided, said method comprising determining the presence of:

a) a Thymine (T) at nucleotide 101 of SEQ ID NO: 1 (SNP_01) or of asequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% sequence identity to SEQ ID NO: 1; and/orb) a Thymine (T) at nucleotide 101 of SEQ ID NO: 2 (SNP_02) or of asequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% sequence identity to SEQ ID NO: 2; and/orc) a molecular marker in between SNP_01 and SNP_02 and which isphysically linked to the RPF12 gene.

In other aspects, the RPF12 gene is detectable using one or more nucleicacid probes, nucleic acid primers, or a combination thereof.

Thus, in one aspect the RPF12 gene is detectable by one or more nucleicacid probes, which hybridize to genomic DNA obtained from a plant orplant part comprising the RPF12 gene using stringent hybridizationconditions.

A nucleic acid probe may for example be a DNA molecule which comprisesSEQ ID NO: 1 (or which comprises a sequence comprising at least 90%,95%, 97%, 98% or more sequence identity to SEQ ID NO: 1) or itscomplement, or SEQ ID NO: 2 (or which comprises a sequence comprising atleast 90%, 95%, 97%, 98% or more sequence identity to SEQ ID NO: 2) orits complement; or a DNA molecule obtained from the genomic sequencebetween SEQ ID NO: 1 (or a sequence comprising at least 90%, 95%, 97%,98% or more sequence identity to SEQ ID NO: 1) and SEQ ID NO:2 (or asequence comprising at least 90%, 95%, 97%, 98% or more sequenceidentity to SEQ ID NO: 2). In another aspect the RPF12 gene isdetectable by one or more nucleic acid primers, which amplify genomicDNA linked to the RPF12 gene. For example, the primers may amplify anucleic acid molecule comprising SNP_01 or SNP_02, or comprising amolecular marker in between SNP_01 and SNP_02. Suitable primers are forexample the 70 to 100 bp upstream and 70 to 100 bp downstream of themarker (e.g. SNP_01 or SNP_02) can be selected to design a forward and areverse primer, which amplify the marker. The primers can be used e.g.for SNP genotyping, e.g. in a KASP-assay for detecting the SNP genotypefor SNP_01 and/or SNP_02.

In any of the probe and/or primer embodiments, or methods of the usingthe same, the probe and/or primer may comprise a label (e.g.,fluorescent label).

In fact, the RPF12 gene (and the Pfs resistance phenotype conferred bythe gene), can be transferred from the seeds deposited under NCIMB42159, or from progeny of said seeds, into any spinach line or varietyby traditional breeding techniques and can confer race 7-14 resistance,optionally further race 1-6 resistance and/or UA4712 resistance (andoptionally resistance against new pathogenic isolates) onto anotherspinach plant. Thus, for example, a spinach plant of the invention canbe used as male or female parent in a cross with another spinach plant,and progeny, such as F1, F2, F3, or further generations of selfingand/or backcross progeny (e.g. BC1, BC2, BC1S1, BC2S1, BC1S2, etc.) canbe identified and selected, whereby the progeny comprise the same Pfsresistance phenotype as the initial plant of the invention. Selection ofprogeny for the presence of the RPF12 gene (and the Pfs resistancephenotype conferred by the gene) can, therefore, be carried out using adisease resistance assay as described herein, whereby resistance againstone or more (or all) of the Pfs races is tested in the progeny.

It is not always necessary to test progeny plants for resistance againstall the Pfs races, as the transfer of resistance against one race isindicative of the transfer of the gene and the resistance against theother races is automatically transferred with the gene. Thus, if thesecond parent in the cross lacks resistance against a particular Pfsrace, then selection of progeny which are resistant against that race issufficient to indicate the transfer of the RPF12 gene.

Therefore, in one aspect of the invention a spinach is providedcomprising resistance against at least Peronospora farinosa races 7-14,optionally further comprising resistance against races 1-6, and/oroptionally further comprising resistance against isolate UA4712 andoptionally new pathogenic isolates. Thus, in one aspect the spinachplant comprises resistance against at least Pfs races 7-14. In anotherembodiment the spinach plant comprises resistance against at least Pfsraces 1-6 and 7-14 (i.e. Pfs 1-14). In a further embodiment the spinachplant comprises resistance against at least Pfs races 7-14 and UA4712and optionally further new pathogenic isolates. In yet a differentembodiment the spinach plant comprises resistance against at least Pfsraces 1-6, 7-14 and UA4712 and optionally further new pathogenicisolates. When reference is made elsewhere herein to ‘resistance againstPfs races 7-14’, or to ‘resistance against at least races Pfs 7-14’, itis understood that the other resistances conferred by the RPF12 gene (asdescribed above) are also encompassed in these or different embodiments.

Whether a spinach plant genotype (i.e. a spinach line or variety)comprises resistance against one or more Pfs races or isolates can betested using qualitative disease resistance assays under controlledenvironment conditions. Different protocols of such assays exist and canbe used by the person skilled in the art. In short, seedlings of aplurality of plants of the plant genotype to be tested (e.g. at least 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) areinoculated with inoculum of the Pfs race and the seedlings are incubatedunder conditions which are favorable to the pathogen. Several days afterincubation, the plants are assessed for infection symptoms, especiallysporulation on the cotyledons and/or leaves (e.g. first true leaf), andeach plant is categorized as “resistant” (showing no signs ofsporulation) or “susceptible” (showing sporulation). If a certainpercentage of all plants of a genotype are classified as “resistant”,e.g. more than about 85%, 90%, 95%, 98%, 99% (or even 100%), then thespinach plant genotype is resistant to the race tested. Obviously, alsoone or more control plants (e.g. a susceptible line or variety, aresistant line or variety) should be included in the assay using thesame treatment(s) and environmental conditions, to ensure that the assayworks as expected.

Alternatively or in addition to the phenotypic assay, selection oridentification of a spinach plant (e.g. a progeny plant) comprising theRPF12 gene of the invention may be achieved by detecting one or moremolecular markers linked to the RPF12 gene or locus, such as SNP_01and/or SNP_02, or a marker located in between SNP_01 and SNP_02 andlinked to the introgressed RPF12 gene. This aspect will be describedelsewhere herein.

In another embodiment, a spinach plant or a progeny of a spinach plantcan be selected for the presence of the RPF12 gene by detecting whetherthe donor genotype for SNP_01 and/or SNP_02 and/or a marker located inbetween SNP_01 and SNP_02 and linked to the introgressed RPF12 gene.

A molecular marker between SNP_01 and SNP_02 linked to the RPF12 gene isa marker (e.g. single nucleotide, SNP, or a sequence of nucleotides)present on the introgression fragment and that has a different genotypethan the cultivated S. oleracea genotype (i.e. is polymorphic with thecultivated spinach). In other words, a marker located in between SNP_01and SNP_02 has a donor genotype, which is the genotype of the wildspinach or wild relative of spinach.

The molecular markers described herein may be detected according to astandard method. For example SNP markers can easily be detected using aKASP-assay (see world wide web at kpbioscience.co.uk) or other SNPgenotyping assays. For example, a TaqMan SNP genotyping assay, a HighResolution Melting (HRM) assay, SNP-genotyping arrays (e.g. Fluidigm,Illumina, etc.) or DNA sequencing may equally be used. For developing aKASP-assay, for example 100 base pairs upstream and 100 base pairsdownstream of the SNP can be selected and two allele-specific forwardprimers and one allele specific reverse primer can be designed. See e.g.Allen et al. 2011, Plant Biotechnology J. 9, 1086-1099, especiallyp097-1098 for KASP assay method. So for example a KASP-assay can bedeveloped easily for SEQ ID NO 1 and/or SEQ ID NO 2 in order to detectthe SNP genotype for SNP_01 and/or SNP_02.

In one embodiment of the invention, the spinach plant is an inbred line,especially an inbred line which can be used as a parent for F1 hybridseed production. In another embodiment of the invention, the spinachplant is a hybrid, especially an F1 hybrid. An F1 hybrid may begenerated by crossing a first inbred parent line which comprises theRPF12 gene, preferably in homozygous form, with a second inbred parentline. In one aspect, the first inbred parent line may be a linedeveloped from using seeds deposited under NCIMB 42159 or from progenyof plants grown from these seeds, whereby the progeny retain the Pfsresistance phenotype (and the RPF12 gene).

In one aspect, the inbred line is a spinach plant of the speciesSpinacia oleracea comprising resistance against Peronospora farinosaraces 7-14, wherein said resistance is conferred by a single dominantgene introgressed from Spinacia tetrandra or Spinacia turkestanica,which gene is located between SNP_01 at nucleotide 101 of SEQ ID NO: 1or of a sequence comprising at least 95% sequence identity to SEQ ID NO:1 and SNP_02 at nucleotide 101 of SEQ ID NO: 2 or of a sequencecomprising at least 95% sequence identity to SEQ ID NO: 2.

The second inbred parent line may be any spinach line, i.e. it maycompletely lack Pfs resistance, or it may comprise a different Pfsresistance gene (and different resistance phenotype) or it may alsocomprise the RPF12 gene.

As the RPF12 gene is dominant, a hybrid spinach plant comprising onlyone copy of the RPF12 gene will show the resistance phenotype conferredby the gene.

As mentioned, the spinach plant according to the invention may be anytype of spinach, as the RPF12 gene can be easily transferred into anyspinach line or variety. For example, the spinach plant may be a savoytype, a semi-savoy type or flat- or smooth leaved spinach. In otherwords, the RPF12 gene can be introduced into any other spinach plant byintrogression from a plant grown from seeds of which a representativesample was deposited under NCIMB 42159, or any spinach plant derivedtherefrom and comprising the RPF12 gene. The deposited seeds aretherefore a source of the RPF12 resistance gene of the invention, as arespinach plants not directly obtained from the deposit, but for exampleindirectly obtained (e.g. later released commercial varieties) and whichcontain the RPF12 gene of the invention. Other sources of the RPF12 genemay be identified, e.g. in wild spinach or wild relatives of spinach ande.g. an allelism test may be used to determine whether another dominantgene, conferring the same Pfs resistance phenotype as the plant of theinvention (or as progeny thereof), is the same gene or a different gene.Alternative methods to determine whether another gene is the same geneinclude the development of molecular markers linked to the RPF12 gene ofthe invention, such as SNP_01 and SNP_02 or any marker in between SNP_01and SNP_02 linked to RPF12, and analyzing whether the markers,especially the donor genotype, also occur in plants comprising the othergene.

The RPF12 gene was identified in wild material from a genebank and wasintroduced through backcrossing into S. oleracea. In one aspect,therefore, a spinach plant is provided comprising resistance against atleast Pfs races 7-14, optionally further against 1-6, and/or optionallyfurther against UA4712 (Pfs race 15) and/or new pathogenic isolates,wherein said resistance against Peronospora farinosa is conferred by anintrogression fragment from wild spinach or from a wild relative ofspinach, wherein the wild relative is selected from Spinacia tetrandaand Spinacia turkestanica.

In one embodiment, the introgression fragment is the fragment as foundin (and as obtainable from; or obtained from; or derivable from; orderived from) spinach seeds, a representative sample of seeds havingbeen deposited under accession number NCIMB 42159. The fragment can beidentified by various methods, such as chromosome painting or sequencingthe spinach genome and identifying chromosome parts which areintrogressions from wild spinach or wild relatives of spinach. Thefragment can also be identified by one or more molecular markers (e.g.SNP markers, AFLP markers, RFLP markers, etc.), especially molecularmarkers which are polymorphic between cultivated spinach and the wildintrogression fragment. Thus, in one aspect the plant comprising theRPF12 gene as found in NCIMB 42159 comprises the same RPF12 markers orgenomic gene sequence or introgression fragment as found in NCIMB42159(or in progeny thereof). In one embodiment, the introgression fragmentcan be identified by determining whether the donor genotype for SNP_01and/or SNP_02 and/or any marker located in between SNP_01 and SNP_02 andbeing linked to RPF12 is present.

In another embodiment, the introgression fragment is derived from thefragment as found in spinach seeds, a representative sample of seedshaving been deposited under accession number NCIMB 42159, whereby theintrogression fragment is shorter but retains the RPF12 gene (and thePfs resistance phenotype conferred by the gene). Spinach plantscomprising such shorter introgression fragments can be generated bycrossing a plant of the invention with another spinach plant andselecting recombinant progeny which retain the resistance phenotypeconferred by the RPF12 gene, but which contain a shorter introgressionfragment. Additionally, SNP_01 and/or SNP_02 and/or any marker locatedin between SNP_01 and SNP_02 and being linked to PRF12 can be used inidentifying recombinant plants comprising a shorter introgressionfragment or sub-fragment of the fragment found in NCIMB42159.

Also provided is a method for generating a spinach plant comprisingresistance against Peronospora farinosa races 7-14 comprises the stepsof:

-   -   a) crossing a first spinach plant of the species Spinacia        oleracea with a second spinach plant which second spinach plant        is susceptible against one or more of Peronospora far/nose races        7 to 14, wherein the first a spinach plant comprising resistance        against Peronospora farinosa races 7-14, wherein said resistance        is conferred by a single dominant gene introgressed from        Spinacia tetrandra or Spinacia turkestanica, which gene is        located between SNP_01 at nucleotide 101 of SEQ ID NO: 1 or of a        sequence comprising at least 95% sequence identity to SEQ ID NO:        1 and SNP_02 at nucleotide 101 of SEQ ID NO: 2 or of a sequence        comprising at least 95% sequence identity to SEQ ID NO: 2;    -   b) selfing a plant grown from progeny of said crossing one or        more times to produce a further generation selfing progeny        and/or backcrossing a plant grown from progeny of said crossing        or grown from the further generation selfing progeny with a        spinach plant is susceptible against one or more of Peronospora        far/nose races 7 to 14; and    -   c) identifying a spinach plant among the progeny plants of        step b) that comprises the a single dominant gene of the first        parent plant of step a).

In one aspect, the genotype of SNP_01 and/or SNP_02 and/or a molecularmarker located in between SNP_01 and SNP_02 (and being linked to theRPF12 gene) is used to identify a plant in step c).

In one aspect, the nucleotide of SNP_01 and/or of SNP_02 is a Thymine,i.e. the donor nucleotide. Therefore in one aspect the plant comprisesan introgression fragment which comprises the donor SNP_01 and/or thedonor SNP_02 genotype.

In another aspect, the genotype for SNP_01 and SNP_02 is the recurrentparent genotype and the introgression fragment is a fragment in betweenSNP_01 and SNP_02. Such a shorter introgression fragment comprises adonor genotype for one or more markers located in between SNP_01 andSNP_02.

A plant produced by the above method is also an embodiment of theinvention.

In one aspect, a method is provided for generating a spinach plantcomprising resistance against at least Pfs races 7-14, optionallyfurther against one or more or all of races 1-6, and/or optionallyagainst UA4712, comprising the steps of:

-   -   a) providing a spinach plant comprising resistance against at        least Pfs races 7-14, optionally further against races one or        more or all of 1-6, and/or optionally against UA4712;    -   b) crossing said spinach plant with another spinach plant to        produce F1 seeds;    -   c) optionally selfing the plants grown from F1 seeds one or more        times to produce F2, F3 or further generation selfing progeny;    -   d) identifying (or selecting) spinach plants grown from F1, F2,        F3 or further generation selfing progeny which have resistance        against at least Pfs races 7-14, optionally further against one        or more or all of races 1-6, and/or optionally against UA4712;    -   e) optionally crossing said identified (or selected) F1, F2, F3        progeny or further generation selfing progeny to the spinach        plant of step b), to produce a backcross progeny; and    -   f) optionally selecting backcross progeny comprising resistance        against at least Pfs races 7-14, optionally further against one        or more or all of races 1-6, and/or optionally against UA4712.

In another embodiment a method for generating a spinach plant comprisingresistance against at least Peronospora farinosa races 7-14 (optionallyfurther against one or more or all of races 1-6, and/or optionallyagainst UA4712) is provided comprises the steps of:

-   -   a) providing a spinach plant comprising an introgression        fragment obtainable from (or as in) accession NCIMB 42159, which        introgression fragment confers resistance against at least        Peronospora farinosa races 7-14 (optionally further against one        or more or all of races 1-6, and/or optionally against UA4712);    -   b) crossing said spinach plant with another spinach plant, for        example with a spinach plant which is susceptible against one or        more of Peronospora farinosa races 7-14, to produce F1 seeds;    -   c) optionally selfing the plants grown from F1 seeds one or more        times to produce F2, F3 or further generation selfing progeny;    -   d) identifying spinach plants grown from F1, F2, F3 or further        generation selfing progeny which have resistance against at        least Peronospora farinosa races 7-14 (optionally further        against races 1-6, and/or optionally against UA4712) and/or        which comprise the introgression fragment or a        resistance-conferring part of the introgression fragment;    -   e) optionally crossing said identified F1 progeny or selfing        progeny to the spinach plant of step b), to produce a backcross        progeny; and    -   f) optionally selecting backcross progeny which comprises        resistance against at least Peronospora farinosa races 7-14        (optionally further against one or more or all of races 1-6,        and/or optionally against UA4712) and/or which comprise the        introgression fragment or a resistance-conferring part of the        introgression fragment.

Regarding the above methods, the following is encompassed herein.

In one aspect, the plant of a) comprises the RPF12 gene as found inseeds deposited under accession number NCIMB 42159. The spinach plantmay be the plant grown from the seeds of the deposit or any spinachplant made using, or having used, the seed deposit and which retains thePfs resistance phenotype (and the gene conferring it). This includescommercial spinach varieties which were made using the seed deposit.Thus, the spinach plant of a) comprises the RPF12 gene according to theinvention, e.g. as found in (or as obtainable from; obtained from;derivable from; derived from) NCIMB 42159. The plant in a) may thereforebe a plant grown from seeds, a representative sample of which has beendeposited under NCIMB42159 (comprising RPF12 in homozygous form) or fromprogeny of such seeds which retain the RPF12 gene and phenotype.

Selections (or identification) in step d) and/or f) may be made based onthe phenotype (i.e. using a Pfs resistance assay) and/or based onmolecular methods, such as detection of molecular markers linked to theRPF12 gene or locus, or other methods such as sequencing. Suitablemarkers are SNP_01, SNP_02 and other markers in between SNP_01 andSNP_02, as described.

In the methods above, the spinach plant of step (a) preferably comprisesthe RPF12 gene (i.e. the introgression fragment comprising the RPF12gene) in homozygous form.

In step b) the spinach plant is, in one aspect, crossed with a spinachplant which is susceptible against at least one of the Pfs races againstwhich the plant of a) is resistant. If the second parent in b) is aspinach plant which is susceptible against at least one of the Pfs racesagainst which the plant of a) is resistant, then the selection in step(d) and/or (f) may be based on selecting plants which now haveresistance against that race.

Steps e) and f) may be repeated one or more times.

In the above methods, plants can also be selected and/or identifiedwhich retain the Pfs resistance phenotype conferred by the RPF12 gene,but which have a smaller introgression fragment. This can haveadvantages, as negative traits coupled to the wild introgressionfragment can thereby be removed. Initial introgession fragments fromwild sources can be quite large, e.g. 20 Mb or 30 Mb. It is thereforepreferred to reduce the size of the introgression fragment byrecombination and to select plants comprising smaller introgressionfragments, but which retain the resistance-conferring part. So, spinachwith all sizes of introgression fragments originating from (or derivedfrom; or derivable from; or obtained from; or obtainable from) seedsdeposited under accession number NCIMB 42159 are included herein, aslong as the Pfs resistance conferring part (i.e. the RPF12 gene) isretained in the spinach plant. As mentioned, the presence can betested/selected phenotypically and/or using molecular methods known inthe art.

Also, a method for identifying, detecting or generating a cultivatedspinach plant or plant part comprising the RPF12 gene is provided,optionally wherein said introgression fragment is as found in NCIMB42159 or a smaller fragment derived therefrom, comprising:

-   -   a) providing a cultivated spinach plant or plant part or DNA of        such plant or plant part,    -   b) screening said plant, plant part or DNA using a molecular        marker assay which detects at least one SNP marker selected from        the group consisting of:        SNP_01, SNP_02, a marker in between SNP_01 and SNP_02 linked to        the RPF12 gene, or a combination thereof; and    -   c) identifying, selecting, and/or generating a plant comprising        the donor genotype for:        SNP_01, SNP_02, a marker in between SNP_01 and SNP_02 linked to        the RPF12 gene, or a combination thereof.

In yet another aspect, a method for detecting whether a cultivatedspinach plant comprises an introgression fragment conferring resistanceagainst at least Pfs races 7-14, and optionally also against isolateUA4712 (Pfs race 15) and/or optionally against one or more of Pfs 1-6 isprovided, said method comprising:

-   -   a) providing cultivated spinach plant or a plant part; and    -   b) screening said plant or said plant part (or DNA obtained from        said plant or plant part) using a molecular marker assay which        detects at least one SNP marker selected from the group        consisting of: SNP_01, SNP_02, a marker in between SNP_01 and        SNP_02 linked to the RPF12 gene, or a combination thereof.

Also plants obtainable or obtained by any of the above methods areembodiments of the invention.

The plants according to the invention may be any cultivated spinach,e.g. savoy, semi-savoy, flat- or smooth leaved spinach. They may beinbred lines, F1 hybrids, double haploids, transgenic plants, mutantplants, a single locus converted plant comprising the RPF12 gene, etc.

Plants of the invention can be used to generate progeny, which have orretain the Pfs resistance phenotype as obtainable from (as present in;as derivable from; as obtained or derived from) seeds deposited underNCIMB 42159. To generate progeny, a spinach according to the inventioncan be selfed and/or crossed one or more times with another spinachplant and seeds can be collected. The presence of the RPF12 gene in theprogeny plants can be determined (i.e. progeny plants comprising theRPF12 gene can be identified/selected) by the Pfs resistance phenotypeand/or molecular methods, such as molecular markers (e.g. SNP markers)closely linked to the RPF12 gene or locus.

Also seeds from which the plants of the invention can be grown areprovided.

In one embodiment, the use of a spinach plant, of which representativeseeds have been deposited under accession number NCIMB 42159, or progenythereof (e.g. obtained by selfing), for generating a spinach plantcomprising Pfs resistance against at least Peronospora farinosa races7-14 (optionally further against races 1-6, and/or optionally againstUA4712) is provided.

In another embodiment, the use of a spinach plant comprising resistanceagainst at least Peronospora farinosa races 7-14 conferred by anintrogression fragment obtainable from (or as present in; as derivablefrom; as obtained or derived from) seeds deposited under accessionnumber NCIMB 42159, or from progeny thereof (e.g. obtained by selfing),for generating spinach plant comprising resistance against at leastPeronospora farinosa races 7-14 (optionally further against races 1-6,and/or optionally against UA4712) is provided.

It is noted that also allelism tests can be used to determine whetherthe resistance gene in a spinach plant is the same gene or a differentgene as the RPF12 gene as present in NCIMB42159 (or in progeny thereof).So, NCIMB42159 (or progeny) can be crossed with another spinach plantcomprising the same resistance phenotype and in progeny of such a crossone can determine in which ratios the phenotype segregates. Thus, in oneaspect a spinach plant is provided comprising resistance against P.ferinosa races 7-14, wherein said resistance gene conferring saidresistance phenotype is the dominant RPF12 gene as present in NCIMB42159(or progeny thereof), i.e. is allelic to RPF12 (is a different allele ofthe RPF12 gene found in NCIMB42159), as determinable in an allelismtest. Allelism tests for dominant genes are known in the art and aree.g. described in Hibberd et al. 1987 (Phytopathology 77: 1304-1307).

Seeds

Seeds from which any of the plants of the invention can be grown areprovided, as are containers or packages containing or comprising suchseeds. Seeds can be distinguished from other seeds due to the presenceof the RPF12 resistance gene, either phenotypically (based on plantshaving the RPF12 resistance phenotype) and/or using molecular methods.Said molecular methods include the use of molecular markers describedherein, i.e. SNP_01, SNP_02, a marker in between SNP_01 and SNP_02linked to the RPF12 gene.

In one aspect, seeds are packaged into small and/or large containers(e.g., bags, cartons, cans, etc.). The seeds may be pelleted prior topacking (to form pills or pellets) and/or treated with variouscompounds, such as seed coatings.

Seed pelleting can be combined with film coating (Halmer, P. 2000.Commercial seed treatment technology. In: Seed technology and itsbiological basis. Eds: Black, M. and Bewley, J. D., pages 257-286).Pelleting creates round or rounded shapes, which are easily sown withmodern sowing machines. A pelleting mixture typically contains seeds andat least glue and filler material. The latter could be, for example,clay, mica, chalk or cellulose. In addition, certain additives can beincluded to improve particular properties of the pellet, e.g., a seedtreatment formulation comprising at least one insecticidal, acaricidal,nematicidal or fungicidal compound can be added directly into thepelleting mixture or in separate layers. A seed treatment formulationcan include one of these types of compounds only, a mixture of two ormore of the same type of compounds or a mixture of one or more of thesame type of compounds with at least one other insecticide, acaricide,nematicide or fungicide.

Formulations especially suitable for the application as a seed treatmentcan be added to the seed in the form of a film coating including alsothe possibility of using the coating in or on a pellet, as well asincluding the seed treatment formulation directly into the pelletmixture. Characteristically, a film coating is a uniform, dust-free,water permeable film, evenly covering the surface of all individualseeds (Halmer, P. 2000. Commercial seed treatment technology. In: Seedtechnology and its biological basis. Eds: Black, M. and Bewley, J. D.,pages 257-286). Besides the formulation, the coating mixture generallyalso contains other ingredients such as water, glue (typically apolymer), filler materials, pigments and certain additives to improveparticular properties of the coating. Several coatings can be combinedon a single seed.

In addition, several combinations with film coating are possible: thefilm coating can be added on the outside of the pellet, in between twolayers of pelleting material, and directly on the seed before thepelleting material is added. Also more than 1 film coating layer can beincorporated in a single pellet. A special type of pelleting isencrusting. This technique uses less filler material, and the result isa ‘mini-pellet’.

Seeds may also be primed. Spinach is often primed. Priming is awater-based process that is performed on seeds to increase uniformity ofgermination and emergence from the soil, and thus enhance vegetablestand establishment. Priming decreases the time span between theemergence of the first and the last seedlings. Methods how to primespinach seeds are well known in the art (see, e.g., Chen et al. 2010,Seed Sci. & Technol. 38: 45-57).

Plant Parts and Vegetative Reproductions

In a further aspect plant parts, obtained from (obtainable from) a plantof the invention are provided herein, and containers or packagescomprising said plant parts.

In a preferred embodiment the plant parts are leaves of spinach plantsof the invention, preferably harvested leaves, or parts of these. Leavesmay be loose, bunched, fresh (e.g. in bags), frozen, blanched or boiled.Leaves may be fresh or processed, they may be part of food or feedproducts, such as salads, etc.

Other plant parts, of plants of the invention, include stems, cuttings,petioles, cotyledons, flowers, anthers, pollen, ovaries, roots, roottips, protoplasts, callus, microspores, stalks, ovules, shoots, seeds,embryos, embryo sacs, cells, meristems, buds etc.

Seeds include for example seeds produced on the plant of the inventionafter self-pollination or seed produced after cross-pollination, e.g.pollination of a plant of the invention with pollen from another spinachplant or pollination of another spinach plant with pollen of a plant ofthe invention.

In one aspect, the plant parts or seeds can be identified by thepresence of the donor genotype for SNP_01 and/or SNP_02 and/or amolecular marker located in between SNP_01 and SNP_02.

In a further aspect, the plant part is a plant cell. In still a furtheraspect, the plant part is a non-regenerable cell or a regenerable cell.A non-regenerable cell is a cell which cannot be regenerated into awhole plant through in vitro culture, but the non-regenerable cell maybe in a plant or plant part (e.g. leaves) of the invention. In anotheraspect, the plant cell is a somatic cell.

In a further aspect the plant cell is a reproductive cell, such as anovule or pollen. These cells are haploid. When they are regenerated intowhole plants, they comprise the haploid genome of the starting plant. Ifchromosome doubling occurs (e.g. through chemical treatment), a doublehaploid plant can be regenerated. In one aspect the plant of theinvention, comprising the RPF12 resistance gene is a haploid or a doublehaploid spinach plant.

Moreover, there is provided an in vitro cell culture or tissue cultureof spinach plants of the invention in which the cell- or tissue cultureis derived from a plant parts described above, such as, for example andwithout limitation, leaves, pollen, embryos, cotyledon, hypocotyls,callus, meristematic cells, roots, root tips, anthers, flowers, seeds orstems, somatic cells, reproductive cells.

Therefore, one aspect provides a cell culture or tissue culturecomprising cells or tissues derived from a part a of a spinach plant ofthe species Spinacia oleracea comprising resistance against Peronosporafarinosa races 7-14, wherein said resistance is conferred by a singledominant gene introgressed from Spinacia tetrandra or Spinaciaturkestanica, which gene is located between SNP_01 at nucleotide 101 ofSEQ ID NO: 1 or of a sequence comprising at least 95% sequence identityto SEQ ID NO: 1 and SNP_02 at nucleotide 101 of SEQ ID NO: 2 or of asequence comprising at least 95% sequence identity to SEQ ID NO: 2.

In one aspect the cells or tissues can be identified by the presence ofthe donor genotype for SNP_01 and/or SNP_02 and/or a molecular markerlocated in between SNP_01 and SNP_02.

Also provided are spinach plants regenerated from the above-describedplant parts, or regenerated from the above-described cell or tissuecultures, said regenerated plant having a Pfs resistance phenotype (asconferred by the RPF12 gene), i.e. retains the RPF12 gene (or theintrogression fragment comprising the RPF12 gene) of the invention.These plants can also be referred to as vegetative propagations ofplants of the invention. In one aspect such plants can be identified bythe presence of the donor genotype for SNP_01 and/or SNP_02 and/or amolecular marker located in between SNP_01 and SNP_02.

Also provided are harvested leaves of plants of the invention andpackages comprising a plurality of leaves of plants of the invention.These leaves thus comprise the RPF12 gene of the invention, detectableby e.g. linked molecular markers or phenotypically (for the originallyused whole plant and/or regenerated plant).

The invention also provides for a food or feed product comprising orconsisting of a plant part described herein. The food or feed productmay be fresh or processed, e.g., canned, steamed, boiled, fried,blanched and/or frozen etc. Examples are salad or salad mixturescomprising leaves or parts of leaves of plants of the invention.

A spinach plant of the invention or a progeny thereof retaining the Pfsresistance phenotype conferred by the RPF12 gene and/or retaining theintrogression fragment comprising the RPF12 gene identifiable by thepresence of the donor genotype for SNP_01 and/or SNP_02 and/or amolecular marker located in between SNP_01 and SNP_02, e.g. as presentin NCIMB 42159, and parts of the afore-mentioned plants, can be suitablypacked for, e.g., transport, and/or sold fresh. Such parts encompass anycells, tissues and organs obtainable from the seedlings or plants, suchas but not limited to: leaves, cuttings, pollen, parts of leaves, andthe like.

Leaves may be harvested immature, as baby-leaf or baby spinach, ormature. A plant, plants or parts thereof may be packed in a container(e.g., bags, cartons, cans, etc.) alone or together with other plants ormaterials. Parts can be stored and/or processed further. Encompassed aretherefore also food or feed products comprising one or more of suchparts, such leaves or parts thereof obtainable from a plant of theinvention, a progeny thereof and parts of the afore-mentioned plants.For example, containers such as cans, boxes, crates, bags, cartons,Modified Atmosphere Packaging, films (e.g. biodegradable films), etc.comprising plant parts of plants (fresh and/or processed) of theinvention are also provided herein.

Plants and Progeny

In another embodiment, plants and parts of spinach plants of theinvention, and progeny of spinach plants of the invention are provided,e.g., grown from seeds, produced by sexual or vegetative reproduction,regenerated from the above-described plant parts, or regenerated fromcell or tissue culture, in which the reproduced (seed propagated orvegetatively propagated) plant comprises resistance against at least Pfsraces 7-14, optionally further against races 1-6, and/or optionallyagainst UA4712 (as conferred by the RPF12 gene).

In one aspect, a progeny plant of a spinach plant of the invention is aprogeny plant that retains the RPF12 resistance gene located betweenSNP_01 of SEQ ID NO 1 and SNP_02 of SEQ ID NO 2 which gene confersresistance to Peronospora farinosa races 7-14.

In another aspect, the progeny plant is a spinach plant of the speciesSpinacia oleracea comprising resistance against Peronospora farinosaraces 7-14, wherein said resistance is conferred by a single dominantgene introgressed from Spinacia tetrandra or Spinacia turkestanica,which gene is located between SNP_01 at nucleotide 101 of SEQ ID NO: 1or of a sequence comprising at least 95% sequence identity to SEQ ID NO:1 and SNP_02 at nucleotide 101 of SEQ ID NO: 2 or of a sequencecomprising at least 95% sequence identity to SEQ ID NO: 2.

Preferably, the presence the RPF12 gene is identifiable by the donorgenotype for SNP_01 and/or SNP_02 and/or a molecular marker located inbetween SNP_01 and SNP_02 and linked to RPF12.

In one aspect, the progeny plant comprises a Thymine for SNP_01 and/orof SNP_02.

As mentioned before, whether or not a plant, progeny or vegetativepropagation comprises the Pfs resistance phenotype as conferred by theRPF12 gene can be tested phenotypically using e.g. the Pfs diseaseresistance assays as described above or in the Examples; and/or usingmolecular techniques such as molecular marker analysis, DNA sequencing(e.g. whole genome sequencing to identify the wild introgression),chromosome painting, etc.

In one embodiment, the RPF12 resistance gene, e.g. as obtainable from(obtained from; as found in) plants deposited under NCIMB 42159, orprogeny thereof, or as obtainable from other wild donors, can becombined with other Peronospora farinosa resistance genes or resistanceloci (e.g. RPF1-RPF6, R6, etc.) or with other traits, such resistanceagainst bacteria (e.g. Pseudomonas syringae pv. spinacea; Erwiniacarotovora), fungi (e.g. Albugo occidentalis; Colletotrichum dematium fsp. spinaciae; Stemphylium botryosum f sp. spinacia), viruses (e.g.viruses causing curly top disease) or nematodes. This can be done bytraditional breeding techniques, e.g. by backcrossing in order tointroduce one or more traits into a plant of the invention or in orderto introduce the RPF12 gene of a plant of the invention into anotherspinach plant comprising such one or more additional traits. Thus, inone aspect a plant of the invention is used as a donor of the RPF12gene, while in another aspect a plant of the invention is used asrecipient of one or more other traits.

Furthermore, the invention provides for progeny comprising or retainingthe Pfs resistance phenotype (conferred by the RPF12 gene), such asprogeny obtained by, e.g., selfing one or more times and/orcross-pollinating a plant of the invention with another spinach plant ofa different variety or breeding line, or with a spinach plant of theinvention one or more times. In particular, the invention provides forprogeny that retain the RPF12 gene (conferring the Pfs resistancephenotype), e.g. of (as found in) NCIMB 42159 or as introgressed fromother wild donor plants. In one aspect the invention provides for aprogeny plant comprising the RPF12 resistance, such as a progeny plantthat is produced from a spinach plant comprising the RPF12 resistance byone or more methods selected from the group consisting of: selfing,crossing, mutation, double haploid production or transformation.

Mutation may be spontaneous mutations or human induced mutations orsomaclonal mutations.

In one embodiment, plants or seeds of the invention may also be mutated(by e.g. irradiation, chemical mutagenesis, heat treatment, TILLING,etc.) and/or mutated seeds or plants may be selected (e.g. naturalvariants, somaclonal variants, etc.) in order to change one or morecharacteristics of the plants. Similarly, plants of the invention may betransformed and regenerated, whereby one or more chimeric genes areintroduced into the plants.

Transformation can be carried out using standard methods, such asAgrobacterium tumefaciens mediated transformation or biolistics,followed by selection of the transformed cells and regeneration intoplants. A desired trait (e.g. genes conferring pest or diseaseresistance, herbicide, fungicide or insecticide tolerance, etc.) can beintroduced into the plants, or progeny thereof, by transforming a plantof the invention or progeny thereof with a transgene that confers thedesired trait, wherein the transformed plant retains the RPF12 gene andthe Pfs resistance phenotype conferred by it and contains the desiredtrait.

The RPF12 gene or allele may be transferred to progeny by furtherbreeding. In one aspect progeny are F₁ progeny obtained by crossing aplant of the invention with another plant or S1 progeny obtained byselfing a plant of the invention. Also encompassed are F2 progenyobtained by selfing the F₁ plants, or further generation progeny.“Further breeding” encompasses traditional breeding techniques (e.g.,selfing, crossing, backcrossing), marker assisted breeding, and/ormutation breeding. In one embodiment, the progeny have the Pfsresistance phenotype of e.g. NCIMB 42159 and comprising the RPF12 gene(or introgression comprising the gene or the reduced size introgressionfragment comprising the RPF12 gene) obtainable/obtained from (or asfound in) e.g. NCIMB 42159 or as introgressed from other wild donorplants.

In one aspect, haploid plants and/or double haploid plants of plant ofthe invention are encompassed herein, which comprise resistance againstat least Peronospora farinosa races 7-14 (optionally further againstraces 1-6, and/or optionally against UA4712), as conferred by the RPF12gene or by the introgression fragment comprising the RPF12 gene. Haploidand double haploid (DH) plants can for example be produced by anther ormicrospore culture and regeneration into a whole plant. For DHproduction chromosome doubling may be induced using known methods, suchas colchicine treatment or the like. So, in one aspect a spinach plantis provided, comprising Pfs resistance phenotype as described, whereinthe plant is a double haploid plant.

In another embodiment, the invention relates to a method for producingspinach seed, comprising crossing a plant of the invention with itselfor a different spinach plant and harvesting the resulting seed. In afurther embodiment the invention relates to seed produced according tothis method and/or a spinach plant produced by growing such seed. Thus,a plant of the invention may be used as male and/or female parent, inthe production of spinach seeds, whereby the plants grown from saidseeds comprise at least Peronospora farinosa races 7-14 (optionallyfurther against races 1-6, and/or optionally against UA4712), due to thepresence of the RPF12 gene.

Thus, in one aspect, progeny of a spinach plant of the invention areprovided, wherein the progeny plant is produced by selfing, crossing,mutation, double haploid production or transformation and wherein theprogeny retain the RPF12 resistance gene (and phenotype conferred by it)described herein, i.e. obtainable by crossing a spinach plant, grownfrom seeds e.g. deposited under accession number NCIMB 42159 or a plantcomprising the RPF12 gene introgressed from another wild donor plant,with another spinach plant. In other words, the resistance gene or locus(or introgression fragment comprising the gene or locus) as presentin/found in/as derived from (or as derivable from) e.g. seed depositNCIMB 42159 or as introgressed from other wild donor plants is retainedin the progeny plants.

Molecular markers may also be used to aid in the identification of theplants (or plant parts or nucleic acids obtained therefrom) containingthe RPF12 resistance gene or locus or allele(s). For example, one candevelop one or more suitable molecular markers which are closelygenetically (and preferably also physically) linked to the RPF12resistance gene, locus or allele. This can be done by crossing aresistant spinach plant (comprising RPF12) with a susceptible spinachplant and developing a segregating population (e.g. F2 or backcrosspopulation) from that cross. The segregating population can then bephenotyped for Pfs resistance and genotyped using e.g. molecular markerssuch as SNPs (Single Nucleotide Polymorphisms), AFLPs (AmplifiedFragment Length Polymorphisms; see, e.g., EP 534 858), or others, and bysoftware analysis molecular markers which co-segregate with the Pfsresistance trait in the segregating population can be identified andtheir order and genetic distance (centimorgan distance, cM) to the RPF12resistance gene or locus can be identified. Molecular markers which areclosely linked to RPF12 resistance locus, e.g. markers at a 5 cMdistance or less, can then be used in detecting and/or selecting plants(e.g. plants of the invention or progeny of a plant of the invention) orplant parts comprising or retaining the introgression fragmentcomprising the RPF12 resistance gene or locus. Such closely linkedmolecular markers can replace phenotypic selection (or be used inaddition to phenotypic selection) in breeding programs, i.e. in MarkerAssisted Selection (MAS). Preferably flanking markers are used in MAS,i.e. one marker on either side of the RPF12 gene or locus, e.g. SNP_01and SNP_02, but also other flanking markers may be used.

Any other type of molecular marker and/or other assay that is able toidentify the relative presence or absence of a trait of interest (i.e.the RPF12 gene or locus) in a plant or plant part can also be useful forbreeding purposes.

DEPOSIT INFORMATION

A total of 2500 seeds of spinach NCIMB 42159 were deposited by NunhemsB.V. on 10 Sep. 2013, at the NCIMB Ltd., Ferguson Building, Craib stoneEstate, Bucksburn, Aberdeen AB21 9YA, United Kingdom (NCIMB). Access tothe deposit will be available during the pendency of this application topersons determined by the Director of the U.S. Patent Office to beentitled thereto upon request. Subject to 37 C.F.R. §1.808(b), allrestrictions imposed by the depositor on the availability to the publicof the deposited material will be irrevocably removed upon the grantingof the patent. The deposit will be maintained for a period of 30 years,or 5 years after the most recent request or for the enforceable life ofthe patent whichever is longer, and will be replaced if it ever becomesnonviable during that period. Applicant does not waive any rightsgranted under this patent on this application or under the Plant VarietyProtection Act (7 USC 2321 et seq.).

Various modifications and variations of the described products andmethods of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in plant breeding,chemistry, biology, plant pathology or related fields are intended to bewithin the scope of the following claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1 Pfs Resistance Phenotype of Spinach Comprising theRPF12 Gene

The resistance to downy mildew infection was assayed with the help of adifferential set obtained from the Naktuinbouw.

Spinach plants of the invention (comprising the RPF12 gene) were plantedalong with spinach plants from different other genotypes (see Table 1)in trays containing BVB substrate (Euroveen, Grubbenvorst), and coveredwith Agra-vermiculite (Pull, Rhenen). Per test at least 10 plants fromone genotype where tested in one or two replications. The trays wereplaced in a climate cell at 12° C./15° C. (day/night) with a 12 hphotoperiod. Plants were inoculated by spraying a sporangial suspension(2.5×10⁵/ml) of a pathogenic race of Peronospora farinosa f sp.spinaciae 14 days after seeding. In this manner, thirteen pathogenicraces were assayed (as shown in Table 2).

The inoculated plants were covered with transparent plastic materialwith 100% relative humidity for a 24 h period, after this period theplastic was removed on top to lower the relative humidity to 80%.

After 10 days, the plants were scored as ‘resistant’ or ‘susceptible’based on symptoms of pathogen sporulation on the cotyledons and trueleaves, as described by Irish et al. (2007; Plant Dis. 91: 1392-1396).Plants exhibiting any evidence of sporulation were considered‘susceptible’. Plants not exhibiting sporulation were considered‘resistant’. Resistant plants were re-inoculated to assess whetherplants initially scored as resistant had escaped infection, or whetherthey were truly resistant. These plants were scored again 10 days afterthe second inoculation.

Any genotype with <15% of plants being categorized as ‘susceptible’(i.e. with >85% of plants categorized as resistant) were considered as aresistant genotype.

Normally the resistance of a spinach hybrid is the effect of two genes,as described by Handke et al. (2000; Gartenbauwissenschaft, 65: 73-78).For example in the hybrid Andromeda, the RPF2 resistance gene (Laziotype, resistance against Pfs races) is combined with a RPF3 (Califlaytype, resistance against Pfs races 1, 3, 5, 8, 9, 11, 12, and 14)resistance gene. This combination of genes results in resistance to Pfsraces 1-12, and 14 and susceptibility to Pfs race 13 (see Table 2). Thenew resistance gene, RPF12, gives full resistance to all currently knownraces. The resistance works both homozygous in fixed lines as inheterozygous backgrounds, like spinach hybrids (see Table 3).

The RPF12 resistance trait of the present invention is conferred by asingle dominant resistance gene, which has the great advantage that theRPF12 resistance trait can be easily transferred into other spinachlines or varieties by crossing/introgression, and that it can easily becombined with other resistance genes. The RPF12 gene confers fullresistance to current Pfs races without the need to select acomplimentary resistance gene.

TABLE 2 Variety Pfs1 Pfs2 Pfs3 Pfs4 Pfs5 Pfs6 Pfs7 Pfs8 Pfs9 Pfs10 Pfs11Pfs12 Pfs13 Pfs14 UA4712 Viroflay + + + + + + + + + + + + + + +Resistoflay − − + + + + + + + + + + + + + Califlay − + − + − + + − − + −− + − + Clermont − − − − + + + + + + + + + + − Campania − − − − − +− + + + − + +/− + − Boeing − − − − − − − + − + − + − + − (=Avenger inUSA) Lion − − − − − − − − − + − − − − − Lazio − − − − − − − − −− + + + + − Whale − − − (−) − (−) (−) − − + − − + (−) + Polka − − − +− + + − − + − − + − + Pigeon − − − − − − − − − − − − − + + R6 − − − − −− + + − + − − − − (data from WO2013/064436*) Andromeda − − − − − − − − −− − − + − − F1 (US2012/0222147) Parent 1 of − + − + − + + − − + − − +− + Andromeda Parent 2 of − − − − − − − − − − + + + + − Andromeda NCIMB− − − − − − − − − − − − − − − 42159 (RPF12)

Table 2 shows the differential set of spinach downy mildew races and theresistance of various spinach varieties (hybrids) to each one of thesepathogenic races. A susceptible reaction (indicated as “+” in the table)is scored when a successful infection by the fungus is visible assporulation on the cotyledon or leaf. Resistance (indicated as “−” inthe table) is the absence of sporulation on the cotyledons and matureleaves. “(−)” indicates reduced level of infection, often referred to asfield resistance=sparse sporulation on the tips of cotyledons. “+/−”indicates variability in the number of resistant and susceptible plantsobserved. ‘nt’ is not tested. *In WO2013/064436 the R6 gene is describedas conferring resistance against Pfs1-6, Pfs9, Pfs11, Pfs12, Pfs13 andPfs14 (UA4410).

Example 2 Introduction of the RPF12 Resistance Trait into Other SpinachPlants

In another experiment, a plant of the invention was crossed (as amother) with a different spinach plant susceptible to all known races.Plants of the F1 population were selfed, and a total of 173 plants ofthe F2 generation were tested for Pfs resistance, as described inExample 1. As a positive discriminator for the presence of the RPF12trait resistance to Pfs races 10, 12, 13, 14, and UA4712 (synonym Pfsrace 15) were assayed, because this resistance was present in the fatherplant (RPF12) but not in the mother plant of the cross.

It was observed that resistance against Pfs races 1, 2, 3, 4, 5, 6, 9,10, 11, 12, 13, 14 and UA4712 (race 15) segregated in the F2 generationin a fashion that corresponds to dominant monogenic inheritance: 130 ofthe 164 F2 plants exhibited the RPF12-resistance phenotype. Table 3gives a detailed overview of the segregation of the RPF12 resistancegene in five Pfs assays. Chi-square tests confirmed that the observedsegregation in the F2 populations was consistent with a 3:1 (resistant:susceptible) segregation of the RPF12 resistance profile.

TABLE 3 Race Outcome Pfs10 Pfs12 Pfs13 Pfs14 UA4712 Total Resistant 21 628 25 50 130 (78%) (75%) (80%) (83%) (78%) (79%) Susceptible 6 2 7 5 1434 (22%) (25%) (20%) (17%) (22%) (21%) Total 27 8 35 30 64 164observations Chi square 0.739 1.000 0.494 0.292 0.564 0.148 probablityFitting the yes yes yes yes yes yes expected segregation pattern(p >0.05) Table 3: Overview of segregation of the RPF12 resistanceprofile in Pfs assays, expressed in the number and percentage of plantsobserved as resistant or susceptible per Pfs race. The plant belong tothe progeny of an inbred from a cross between a spinach plant whichlacks the RPF12 gene and a genotype of the invention containing theRPF12 gene. Chi-square tests confirm that the results fit the expectedsegregation pattern for a dominant monogenic trait. In all caseschi-square values are well above 0.05.

Example 3 Mapping of Resistance Gene RPF12

A F2 population was developed by crossing spinach plants of theinvention, containing RPF12, with a spinach plant that does not containthe RPF12 resistance gene. Linkage mapping was conducted, and SingleNucleotide Polymorphism markers (SNPs) SNP_01 and SNP_02 shown in Table4 were identified flanking the RPF12 gene.

TABLE 4 SNP markers flanking the RPF12 gene SNP geno- SNP geno-SNP geno- type type type in spinach in spinach of the plant com-plant com- recurrent SNP and prising the prising the parent, nucleotidedonor donor lacking the position fragment fragment intro- (nt) in thein homo- in hetero- gression sequence ygous form ygous form fragmentSNP marker sequence SNP_01 at TT TC CC AAGGAAAAAAGAGAGCAT nt 101GATCTTAACATGTAATTAG CTTAGAGATGAGTAAACG ACTAAATGTGTATAATCATACGCTATACTGCTATAGTG TTACATA[C/T]AAGAATAG TGTCCACTTTATAAAGTGATCGGTAACAGTTATATTTT CTAGTCGAGCCAGATCAA CCATGAATATGTTTGGGAGTTGCTCAAGCGTTAGAT (SEQ ID NO: 1) SNP_02 at TT TC CC ATTGGTGGTAAACGAGTAnt 101 AGACAACATATATTTGAA CCTTCTCAAACTTCGTTTT AACAGGACTAATAATATTGCATTATCATTGTTATTTT ATCATACT[C/T]CGTACAA TGTGAATTCGAAATAAACAACTATAGGATTCGAATT ACTACATGTTAATTGTTGA AACCAAATATTACTCACATAGTCACATATTCACATGTG (SEQ ID NO: 2)

1. A spinach plant of the species Spinacia oleracea comprisingresistance against Peronospora farinosa races 7-14, wherein saidresistance is conferred by a single dominant gene, wherein said gene islocated between SNP_01 at nucleotide 101 of SEQ ID NO: 1 or of asequence comprising at least 95% sequence identity to SEQ ID NO: 1 andSNP_02 at nucleotide 101 of SEQ ID NO: 2 or of a sequence comprising atleast 95% sequence identity to SEQ ID NO:
 2. 2. The plant according toclaim 1, wherein the gene further confers resistance against Peronosporafarinosa races 1, 2, 3, 4, 5 and
 6. 3. The plant according to claim 1,wherein the gene further confers resistance against Peronospora farinosarace
 15. 4. The spinach plant according to claim 1, wherein thenucleotide of SNP_01 and/or of SNP_02 is a Thymine.
 5. The plantaccording to claim 1, wherein said resistance gene is the gene presentin plants grown from seeds, a representative sample of seeds having beendeposited under accession number NCIMB
 42159. 6. The plant according toclaim 1, wherein said spinach plant is a hybrid plant.
 7. The plantaccording to claim 1, wherein said spinach plant is an inbred plant. 8.The plant according to claim 1, wherein the spinach plant is savoy,semi-savoy, flat- or smooth leaved.
 9. The plant according to claim 1,wherein said resistance against Peronospora farinosa is conferred by anintrogression fragment from Spinacia turkestanica.
 10. The plantaccording to claim 9, wherein said introgression fragment is thefragment as found in spinach seeds, a representative sample of seedshaving been deposited under accession number NCIMB 42159, or asub-fragment thereof.
 11. A seed from which a plant according to claim 1can be grown.
 12. A leaf of a plant according claim
 1. 13. A progenyplant of a spinach plant according to claim 1, wherein said progenyplant retains the resistance gene located between SNP_01 of SEQ ID NO: 1and SNP_02 of SEQ ID NO: 2 which gene confers resistance to Peronosporafarinosa races 7-14.
 14. The progeny plant according to claim 13,wherein said progeny plant is resistant against Peronospora farinosaraces 7-14 and race
 15. 15. The progeny plant according to claim 13,wherein the progeny plant is produced by one or more of selfing,crossing, mutation, double haploid production and/or transformation. 16.The progeny plant according to claim 13, wherein the nucleotide ofSNP_01 and/or of SNP_02 is a Thymine.
 17. A part of a spinach plantaccording to claim 1, wherein the part is selected from the groupconsisting of: a stem, a cutting, a petiole, a cotyledon, a flower, ananther, a pollen, an ovary, a root, a root tip, a protoplast, a callus,a microspore, a stalk, an ovule, a shoot, a seed, an embryo, an embryosac, a cell, a meristem, a bud and a leaf.
 18. A cell culture or tissueculture comprising cells or tissues derived from a part a of a spinachplant of the species Spinacia oleracea comprising resistance againstPeronospora farinosa races 7-14, wherein said resistance is conferred bya single dominant gene introgressed from Spinacia tetrandra or Spinaciaturkestanica, which gene is located between SNP_01 at nucleotide 101 ofSEQ ID NO: 1 or of a sequence comprising at least 95% sequence identityto SEQ ID NO: 1 and SNP_02 at nucleotide 101 of SEQ ID NO: 2 or of asequence comprising at least 95% sequence identity to SEQ ID NO:
 2. 19.A spinach plant regenerated from the cell culture or tissue culture ofclaim 19 and comprising resistance against Peronospora farinosa races7-14.
 20. A method for generating a spinach plant comprising resistanceagainst Peronospora farinosa races 7-14 comprising: a) crossing a firstspinach plant of the species Spinacia oleracea that comprises resistanceagainst Peronospora farinosa races 7-14 with a second spinach plant thatis susceptible against one or more of Peronospora farinosa races 7 to14, wherein said resistance is conferred by a single dominant gene, andwherein said gene is located between SNP_01 at nucleotide 101 of SEQ IDNO: 1 or of a sequence comprising at least 95% sequence identity to SEQID NO: 1 and SNP_02 at nucleotide 101 of SEQ ID NO: 2 or of a sequencecomprising at least 95% sequence identity to SEQ ID NO: 2; and b)growing a progeny plant from said crossing, wherein said progeny plantcomprises said single gene.
 21. The method of claim 20, furthercomprising c) selfing a plant grown from progeny of said crossing, oneor more times to produce a further generation selfing progeny and/orbackcrossing a plant grown from progeny of said crossing or grown fromthe further generation selfing progeny with a spinach plant issusceptible against one or more of Peronospora far/nose races 7 to 14;and d) identifying a spinach plant among the progeny plants of step c)that comprises said single dominant gene of the first parent plant ofstep a).
 22. A spinach plant obtained by the method of claim 21, saidspinach plant comprising resistance against Peronospora farinosa races7-14.
 23. The method according to claim 21, wherein the nucleotide ofSNP_01 and/or of SNP_02 is a Thymine.
 24. The method according to claim21, wherein the genotype of SNP_01, SNP_02 or a molecular marker locatedin between SNP_01 and SNP_02 is used to identify a plant in step d).